Proteins containing non-natural amino acids hold great promise for biomedical and therapeutic purposes. Such amino acids may be particularly useful in the structural and functional probing of proteins, construction of peptide libraries for combinatorial chemistry, and in proteomics. However, the synthesis of such proteins has been exceptionally costly and inefficient. In the translation system that is known to occur currently in nature, part of the genetic coding mechanism is carried out by aminoacyl-tRNA synthetases (ARSs), which are proteins that charge amino acids onto their cognate tRNAs such that the amino acids are incorporated correctly into growing polypeptide chains by the translational machinery.
ARSs exist in 20 different forms, each of which specifically catalyzes the esterification of a single amino acid to its cognate tRNA isoacceptor, thereby directly connecting the amino acid with its corresponding anticodon triplet. Because mis-charging of noncognate amino acids to tRNAs causes misincorporation of amino acids into cellular proteins which can be fatal to their intracellular activity, the fidelity of the aminoacylation reactions by the ARSs must be extremely high. To achieve this important task, the ARSs use very sophisticated mechanisms to selectively recognize the cognate amino acids and tRNAs. The recognition determinants of tRNAs are diverse ranging from the anticodon loop to the acceptor-stem and the phosphate-ribose backbone. Because of these complexities, engineering of ARSs with desired specificities toward non-natural tRNAs and amino acids has not been achieved. As a result, attention has turned to nucleic acids.
For many years, nucleic acids were considered to be only informational molecules. However, the pioneering work of Cech and coworkers (Cech, 1987, Science, 236:1532-1539; McCorkle et al., 1987, Concepts Biochem. 64:221-226) demonstrated the presence of naturally occurring RNAs that can act as catalysts (ribozymes). However, although these natural RNA catalysts have only been shown to act on ribonucleic acid substrates for cleavage and splicing, the recent development of artificial evolution of ribozymes has expanded the repertoire of catalysis to various chemical reactions. For example, RNAs have been reported to catalyze phosphodiester cleavage on DNA (Beaudry et al., 1992, Science, 257:635), hydrolysis of aminoacyl esters (Piccirilli et al., 1992, Science, 256:1420-1424), self-cleavage (Pan et al., 1992, Biochemistry, 31:3887), ligation of an oligonucleotide with a 3′OH to the 5′triphosphate end of the catalyst (Bartel et al., 1993, Science, 261:14111418), biphenyl isomerase activity (Schultz et al., 1994, Science, 264:1924-1927), and polynucleotide kinase activity (Lorsch et al., 1994, Nature, 371:31-36).
To identify novel catalysts, Brennen et al. (1992, Proc. Natl. Acad. Sci., U.S.A, 89:5381-5383) constructed a heterogeneous pool of macromolecules and used an in vitro selection process to isolate molecules that catalyze the desired reaction. A variation of this approach has been used by Gold et al. (U.S. Pat. No. 5,475,096). This method, known as Systematic Evolution of Ligands by Exponential enrichment (SELEX), identifies nucleic acids that have the ability to form specific, non-covalent interactions with a variety of target molecules. A related patent (U.S. Pat. No. 5,990,142) is based on the SELEX method, but can potentially identify modified and non-modified RNA molecules that can catalyze covalent bond formation with a target. Recently, a similar approach was used to identify catalytic RNA molecules having phosphodiesterase and amidase activity (U.S. Pat. No. 6,063,566 to Joyce).
Additionally, studies have identified RNA molecules that can catalyze aminoacyl-RNA bonds on their own (2′)3′-termini (Illangakekare et al., 1995 Science 267:643-647), and an RNA molecule which can transfer an amino acid from one RNA molecule to another (Lohse et al., 1996, Nature 381:442-444). The predominant method for the in vitro synthesis of aminoacyl-tRNAs currently relies on chemical aminoacylation of a dinucleotide followed by enzymatic ligation to an engineered tRNA fragment. These steps are unfortunately time consuming and laborious (Heckler et al., Biochemistry, 1984 (23) 1468-1473). Thus, there is a need for a method for the aminoacylation of tRNAs wherein any tRNAs of choice can be aminoacylated with desired amino.